Ehrlichia chaffeensis (Ech) is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis (HME), an emerging life-threatening infectious disease in the US. Based on our discoveries of a novel Ech component and host signaling pathways required for obligatory intracellular infection viz. Ech survival, our objective is to develop new compounds and techniques to further elucidate molecular mechanisms and treat obligatory intracellular bacterial infection. We showed that Ech has a unique type IV secretion system effector; Ehrlichial translocated factor-1 (Etf-1), which is abundantly produced and secreted in infected cells. Etf-1 is critical for Ech infection of human monocytes. We demonstrated that Etf-1 subverts and manipulates two important innate immune defense mechanisms against intracellular infection, cellular apoptosis and autophagy. Thus, Etf-1 is a potential target for anti-Ech therapy. Major barriers to Ech therapy and research progress are that 1) the important disease-associated Ech molecules are intracellular, protected from direct antibody or drug attack, and 2) protein-protein interactions (PPIs) such as those induced by Etf-1 are examples of ?undruggable? targets because small molecule inhibitors generally do not bind to the PPI interfaces with high affinity or specificity. However, cyclic peptides (cyclic Ps) are very effective inhibitors of PPIs and because cyclic Ps are proteolytically stable, non-immunogenic, and biologically active in cellular assays, they have great potential as Ech therapeutic agents and research tools. In collaboration with cyclic P expert co-I, we propose an innovative approach: to develop a cell-permeable bicyclic peptides (bicyclic Ps) as synthetic Ech inhibitors. We will synthesize a combinatorial library of ~10 million bicyclic Ps by fusing the first ring featuring a degenerate sequence and the second ring of a novel invariant cyclic cell-penetrating peptide. Our hypothesis is that bicyclic Ps are effective molecular tools and therapeutic agents to block Etf-1 functions and Ech infection. To test our hypothesis, in Aim 1, we will generate high-throughput screen random libraries of cell-permeable bicyclic Ps for Etf-1 binding and inhibition of Ech infection by: 1) synthesizing large one-bead-one-compound random libraries of bicyclic Ps and screening the libraries for binding to Etf-1; 2) sequencing, and chemically resynthesizing high affinity Etf-1-binding bicyclic Ps in sufficient quantity to screen for neutralization of Ech infection in host cells. In Aim 2, we will investigate the mechanism by which bicyclic Ps inhibit Etf-1 and Ech infection by examining: 1) bicyclic P blockade of Etf-1 translocation to mitochondria and inhibition of cellular apoptosis by Etf-1, 2) bicyclic P inhibition of Etf-1-induced cellular autophagy, Etf-1 activation of class III PtdIns3K, and formation of the Etf-1-BECN1-VPS34-RAB5 complex; and 3) efficacy of inhibition of Ech infection by selected combinations of bicyclic Ps. Our results will demonstrate the innovative use of a technology for intracellular delivery of compounds that interfere with targeted PPIs. This approach will overcome current barriers and advance research on obligatory intracellular bacterial infection.